Transmission Electron Microscopy as the Visualization Technique for Analysis of Circadian Synaptic Plasticity in the Mouse Barrel Cortex

Summary

The presented protocol describes the use of transmission electron microscopy (TEM) to quantify circadian changes in the mouse barrel cortex, mainly focusing on synapse number and dendritic spine morphology.

Abstract

Examining circadian synaptic plasticity requires housing mice under different lighting conditions (light/dark cycle, LD 12:12, and constant darkness, DD), providing access to running wheels, and sacrificing them at four defined time points within 24 h-at the beginning and middle of the day/subjective day and at the beginning and middle of the night/subjective night. Brains are then properly fixed for transmission electron microscopy (TEM). The barrel cortex, with its precise somatotopic organization, provides an ideal model for such analysis. To obtain the required brain area, the brains are tangentially cut with a vibratome, and then, sections containing the barrel cortex are selected and embedded in Polybed resin. From the prepared blocks containing the selected barrels, consecutive ultrathin sections are cut. Synaptic density, excitatory and inhibitory, is analysed from electron micrographs using the stereological dissector method. Additionally, TEM images are used for 3D reconstructions of dendritic spines. Changes in the shape of dendritic spines indicate remodeling of neurons during the day. The number of excitatory synapses peaks during sleep (day) in mice, while inhibitory synapses peak during their activity phase (in the middle of the night).

Introduction

Circadian rhythms are generated by circadian clocks in almost all processes in an organism. In animals and humans, they have been detected at molecular, cellular, and whole-organism levels, as well as in their behavior. The circadian system of an organism consists of the main circadian clock (pacemaker) and peripheral clocks. All circadian clocks generate circadian oscillations through the cyclic expression of clock genes, which are controlled by their proteins. The molecular mechanism of the clock generates circadian rhythms with a period of ~1 day (longer or shorter than 24 h), but under day/night conditions, the period of endogenous rhythms is synchronized to 24 h.....

Protocol

All experimental procedures involving animals were approved by the appropriate institutional ethics committee and conducted in accordance with Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes, as well as with national regulations. All efforts were made to minimize animal suffering and to reduce the number of animals used.

1. Preparation of brain tissues

1. Sacrifice mice at four different time points every 6 h over a 24 h cycle, in both light/dark (LD 12:12) and constant darkness (DD) conditions (Figure 1). In ....

Results

To properly apply our method to the analysis of circadian synaptic changes, it is necessary to begin by selecting at least four time points at equal intervals, with two points for each phase of the animals' activity (every 6 h). At these designated time points, the animals are sacrificed, and their brains are collected. This approach allows for the identification of daily or circadian patterns of synaptic plasticity and links them to changes in the animals' locomotor activity (Figure 1). Usi.......

Discussion

Here, we presented the methodology used for studying the circadian plasticity of synapses and the reconstruction of dendritic spines in the barrel cortex of mice. To ensure reliable results, the circadian plasticity study should include at least four time points. Our research showed that data from two time points -- one during the rest phase (day) and one during the activity phase (night) -- provided information on the daily differences between the activity phases of animals under LD 12:12 conditions. Total synapse densi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aclar film	Agar scientific	AGL4458
Blender software			version: v. 2.91.2
Cacodylic acid, sodium salt trihydrate	Polysciences	01131-100
Concave slide	Menzel	9.161 151
Diamond knife	Diatome	15-US	Knife angle: Ultra 45°
Digital camera	Nikon	DXM 1200 F
di-Sodium Hydrogen Phosphate Dodecahydrate	POCH	799280115
Embedding resin	Polysciences	08792-1	Luft formulation
Ethanol	Pol-Aura	PA-11-0004
GIMP software			version: v 3.0.4
Lead citrate	TAAB	L018
Light microscopy	Nikon	Optiphot
Osmium tetroxide	Polysciences	0223C-10	Crystalline (99.95%)
Paintbrush
Photoshop software			version: CS5
Potassium ferricyanide	Sigmaaldrich	P8131
Propylene oxide	Sigmaaldrich	82320	puriss. p.a., ≥99.5% (GC)
Single slot grids	Agar scientific	AGG2525	2 x 0.75 mm or 2 x 1 mm
Sodium chloride	Chempur	117941206
Sodium dihydrogen phosphate dihydrate	Chempur	117991808
Stereo microscope	pzo
Syring filter	Biosens	BS25PES045
TEM	Joel	JEM-2100
Ultramicrotome	Leica	UC7
Uranyl acetate	Lachema
Vibratome	Leica	VT1000 S